TISSUE-SPECIFIC STEM CELLS

Life-Sparing Effect of Human Cord Blood-Mesenchymal Stem Cells in Experimental Acute Kidney Injury
MARINA MORIGI,a CINZIA ROTA,a TIZIANA MONTEMURRO,b ELISA MONTELATICI,b VIVIANA LO CICERO,b BARBARA IMBERTI,a MAURO ABBATE,a CARLA ZOJA,a PAOLA CASSIS,a LORENA LONGARETTI,a PAOLO REBULLA,b MARTINO INTRONA,c CHIARA CAPELLI,c ARIELA BENIGNI,a GIUSEPPE REMUZZI,a,c LORENZA LAZZARIb Mario Negri Institute for Pharmacological Research, Bergamo, Italy; bCell Factory, Centro di Medicina Trasfusionale, Terapia Cellulare e Criobiologia, Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milan, Italy; cLaboratory of Cell Therapy G. Lanzani, Unit of Hematology and Unit of Nephrology and Dialysis, Azienda Ospedaliera, Ospedali Riuniti di Bergamo, Bergamo, Italy
a

Key Words. Human cord blood-mesenchymal stem cells Acute renal failure Tubular cells Kidney repair

ABSTRACT
In search for new sources of mesenchymal stem cells (MSCs) for renal repair in acute kidney injury (AKI), we investigated the potential of human cord blood (CB)-MSCs to cure mice with AKI. Infusion of CB-MSCs in immunodecient mice with cisplatin-induced AKI ameliorated both renal function and tubular cell injury, and prolonged survival. Transplanted CB-MSCs localized in peritubular areas, limited capillary alterations and neutrophil inltration. Apoptosis reduced and tubular cell proliferation increased by virtue of stem cell capacity to produce growth factors. The reno-protective effect of CB-MSCs was further conrmed by their ability to inhibit oxidative damage and to induce the prosurvival factor Akt in tubular cells. The evidence that CB-MSCs in vitro increased the production of growth factors and inhibited IL-1b and TNFa synthesis when cocultured with damaged proximal tubular cells indicates a regenerative and anti-inammatory action of stem cell treatment. Altogether these results highlight the potential of human CB-MSCs as future cell therapy for testing in human AKI. STEM CELLS 2010;28:513522

Disclosure of potential conicts of interest is found at the end of this article.

INTRODUCTION
New hopes for curing acutely damaged organs come from stem cell-based treatment exploiting stem cells peculiar properties of tropism and regenerative capability [17]. In this context, mesenchymal stem cells (MSCs) represent an attractive tool because of their benecial effects on tissue repair in experimental models of myocardial infarction [4, 8, 9], neurologic disease [3, 10], and more recently, in experimental acute kidney injury (AKI) [1114]. MSCs were originally isolated from bone marrow (BM) [1518], but similar cell populations were obtained from other tissues including umbilical cord blood (CB), adipose tissue, peripheral blood, dermal connective tissue, and skeletal muscle [1822]. Studies by our group

documented that murine [11, 12] and human (h) [13] MSCs of BM origin infused in mice with cisplatin-induced AKI promoted renal functional and structural recovery by stimulating renal resident cells to proliferate via local release of the growth factor insulin like growth factor-1 (IGF-1) [12]. Consistently, in the ischemia/reperfusion rat model, MSCs exerted a protective effect via paracrine production of mitogenic, antiapoptotic, and vasculotropic factors [23, 24]. Although BM represents the most common tissue source of MSCs, harvesting these stem cells is invasive and their number, differentiation potential, and life span decline with age. Therefore, the search for new sources of MSCs in the clinical perspective to cure AKI is of signicant value. AKI [2527] is a disease with extremely serious outcome. The mortality associated with this syndrome has remained unchanged worldwide

Author contributions: M.M.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; C.R.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; T.M.: provision of study material, collection and/or assembly of data; E.M.: provision of study material, collection and/or assembly of data; V.L.C.: provision of study material, collection and/or assembly of data; B.I.: collection and/or assembly of data, data analysis and interpretation, manuscript writing; M.A.: collection and/or assembly of data, data analysis and interpretation, manuscript writing; C.Z.: collection and/or assembly of data, data analysis and interpretation; P.C.: collection and/or assembly of data; L. Longaretti: collection and/or assembly of data; P.R.: provision of study material, collection and/or assembly of data, data analysis and interpretation, nal approval of manuscript; M.I.: provision of study material and data analysis and interpretation; C.C.: provision of study material; A.B.: conception and design, data analysis and interpretation, manuscript writing, nal approval of manuscript; G.R.: conception and design, data analysis and interpretation, manuscript writing, nal approval of manuscript; L. Lazzari: provision of study material, collection and/ or assembly of data, data analysis and interpretation, nal approval of manuscript. M.M. and C.R. contributed equally to this article. Correspondence: Marina Morigi, Ph.D., Mario Negri Institute for Pharmacological Research, Via Gavazzeni 11, 24125 Bergamo, Italy. Telephone: 39-035-319-888; Fax: 39-035-319-331; e-mail: morigi@marionegri.it Received June 10, 2009; accepted for publiC cation December 22, 2009; rst published online in STEM CELLS EXPRESS January 4, 2010. V AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.293

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during the past 30 years [25]. To accelerate renal regeneration and to improve survival, several pharmacological therapies have been approached. However, these strategies, while working experimentally, failed when applied to patients. Recent studies have identied CB as an alternative reservoir, readily available and extremely rich of stem and progenitor cells [19, 21, 2830]. The extensive characterization of human (h) CB-MSCs has indicated that they are similar to MSCs of BM origin with respect to morphological characteristics and multipotency [3133]. As for the gene expression prole, the two MSC lineages share all of the most expressed transcripts, but hCB-MSCs express higher levels of genes involved in matrix remodeling and angiogenesis [32]. hCB-MSCs also deserve attention for their capability to synthesize growth factors [19, 33, 34] with anti-apoptotic and regenerative effects on renal tubular cells, the key target of AKI. Early clinical evidence documented both safety and efcacy of CB progenitor cell infusion in humans for the treatment of hematological [22, 35, 36], neurological disorders [21, 37], and Buergers disease [38]. Altogether these ndings prompted us to investigate whether hCB-MSCs could represent a new option to cure AKI. Here, we evaluated whether hCB-MSC treatment could oppose the course of AKI in cisplatin-treated immunodecient mice and by which mechanisms this could occur.

coulter.com). Cells analyzed at passage 3 (data not shown) and 6 showed similar cell surface antigen prole. Cells were incubated with conjugated mouse-anti-human antibodies: CD65-PE-Cy7 (phycoerythrin-Cy7; Beckman Coulter), CD34-PE (phycoerythrin; ` Becton Dickinson, San Jose, CA), CD146-FITC (uorescein isocyanate; Biocytex, Marseille, France), CD90-PE (Chemicon, Temecula, CA, http://www.chemicon.com), CD44-FITC (Becton Dickinson), VE-Cadherin-FITC (Bender MedSystems, Burlingame, CA), alpha SMA-FITC (Sigma-Aldrich), NG2-PE (Beckman Coulter), CD105-PE (Biocytex), CD73-PE (Becton Dickinson), CXCR4-APC (allophycocyanin; Becton Dickinson), CD56PE (Cymbus, Hampshire, U.K.), LNGFR-PE (Becton Dickinson), HLA: human leukocyte antigen class I-FITC (Immunotech Beckman Coulter Company, Praha, Czech Republic), and HLA class II (Becton Dickinson). Additional samples were stained with the primary unconjugated mouse anti-human antibodies: CK3/12 (Biodesign, Maine, USA), CK19 (Biodesign), BB9 (Becton Dickinson), and SSEA4: stage-specic embryonic antigen 4 (Chemicon) followed by FITC-conjugated and PE-conjugated goat anti-mouse antibodies (Exalpha, Maynard, MA). Isotype immunoglobulins IgG1 PE-FITC (Chemicon), IgG1 PC7 (Beckman Coulter), and IgG1 APC (Becton Dickinson) were used as negative controls. Cells were permeabilized with 0.1% Triton X-100 (Sigma) as appropriate.

In Vivo Experiments
Female 2-month-old NOD-SCID mice (Charles River Italia s.p.a., Calco, Italy) were used. Animal care and treatment were conducted in conformity with the institutional guidelines and international laws and policies. AKI was set up in NOD-SCID mice by subcutaneous injection of the nephrotoxic drug cisplatin (SigmaAldrich) at the dose of 12.7 mg/kg as previously described [13]. To investigate the effect of hCB-MSCs in cisplatin-induced AKI model, mice were divided into two groups and intravenously (i.v.) injected 24 hours after cisplatin as follows: group 1, saline (n 12); group 2, hCB-MSCs at sixth passage (5 105 cells per mouse; n 11). Mice were sacriced at 4 days after cisplatin and kidney samples were used for histology and immunohistochemistry evaluations. In another experiment, seven animals for each group were used for assessment of survival. Renal function was assessed as blood urea nitrogen (BUN) by the Reotron test (Roche Diagnostics Corporation, Indianapolis, USA). BUN levels exceeding 30 mg/dl were considered abnormal. The effect of human broblasts infused in cisplatinNOD-SCID mice has been previously reported [13]. By additional experiments, we compared the effect of hCBMSCs with that of human bone marrow (hBM)-MSCs on renal function (at 4 days) and animal survival. hBM-MSCs were isolated as previously described [13, 39]. Mice were divided into three groups and i.v. injected 24 hours after cisplatin as follows: group 1, saline (n 8); group 2, hCB-MSCs (5 105 cells per mouse; n 10); group 3, hBM-MSCs (5 105 cells per mouse; n 10).

MATERIALS

AND

METHODS

Isolation and Culture of hCB-MSCs

Human CB was collected after informed consent of the mother from full-term newborns and isolation of MSCs was performed. Briey, within 12 hours, CB was centrifuged, plasma discarded, and an enrichment protocol was performed by using Rosette Sep enrichment cocktail (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com). The blood was diluted, laid on density gradient Lympholyte-H (1.077 g/ml; Cedarlane, Hornby, ON, Canada, http://www.cedarlanelabs.com), and following centrifugation, the low-density cell fraction was collected. After washing, cells were seeded at 1 106 cells per square centimeter in the presence of a-Modied Eagle Medium (a-MEM; Invitrogen, Carlsdad, CA, http://www.invitrogen.com) supplemented with 20% FBS (Biochrom AG, Berlin, Germany, http:// www.biochrom.de), 2 mM L-glutammine, and antibiotics. Medium was changed every 23 days, cells were detached using trypsin 0.25% EDTA when subconuent (80%) and split 1:3. This technique allows to reach a 54% of isolation efciency.

Cell Lineage Differentiation

Differentiation potential of hCB-MSCs was investigated at passage 6. To induce adipogenic differentiation, at conuence hCB-MSCs were incubated with hMSC Adipogenic Induction and Maintenance Media (Cambrex, Walkersville, MD, http:// www.cambrex.com) replaced every 23 days, for 3 weeks. Oil Red O staining (Sigma-Aldrich, St. Louis, MO, http://www. sigmaaldrich.com) was used to detect lipid vacuoles. For osteogenic differentiation hCB-MSCs were grown for 23 weeks with hMSC Osteogenic Medium (Cambrex) replaced every 34 days. Calcium deposits were stained with Alizarin Red (SigmaAldrich). Chondrocyte differentiation was evaluated in hCBMSCs centrifuged to form a pellet and cultured in differentiation chondrogenic medium (0.5 ml; Cambrex) with TGFb (10 ng/ml) for 2 weeks. Cell clump was xed in paraformaldehyde (Sigma) and stained with Alcian Blue (Sigma-Aldrich) to detect proteoglycans.

Renal Morphology
Renal Histology. Kidney samples were xed in Duboscq-Brazil and parafn-sections were stained with hematoxylin and eosin or periodic acid-Schiffs reagent (PAS). Luminal hyaline casts and tubular necrosis (denudation of tubular basement membrane) were assessed in nonoverlapping elds (up to 28 for each section) using a 40 objective (high power eld [HPF]). Number of casts and tubular proles showing necrosis were analyzed twice in a single-blind fashion. Kidney samples were also taken from mice (n 3) which survived 40 days after hCB-MSC injection to assess the possible presence of hCB-MSC maldifferentiation as previously documented for rat BM-MSCs in other animal models [40]. Samples were processed as described earlier. Electron Microscopy.
Fragments of kidney tissue were xed for 4 hours in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, and washed repeatedly in the same buffer. After

Flow Cytometry Analysis

hCB-MSCs were characterized by ow cytometry (Cytomics FC500, Beckman Coulter, Fullerton, CA, http://www.beckman-

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postxation in 1% OsO4, specimens were dehydrated through ascending grades of alcohol and embedded in Epon resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a Philips Morgagni electron microscope.

ABC kit (Vector Laboratories) using DAB as substrate. Nuclei were counterstained with Harris hematoxylin. Evaluation was performed by counting PCNA positive cells in tubular proles in at least 15 HPF ( 400)/section (n 6 mice for each group).

Quantication of PKH-26-Labeled hCB-MSCs

To study intrarenal localization of hCB-MSCs, cells labeled with PKH-26 red uorescence cell linker (Sigma-Aldrich) were infused in mice 24 hours after cisplatin. Labeling efcacy was assessed to be >98%. Viability evaluated by Trypan blue exclusion was >96%. At 4 days, mice were sacriced and kidney samples were xed in 4% paraformaldehyde and sections stained with FITC-labeled lectin Wheat Germ Agglutinin (WGA; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com), 40 ,60 -diamidino-2phenylindole dihydrochloride hydrate (DAPI; Sigma-Aldrich) as previously reported [13]. Slides were analyzed for PKH-26 positive cells. A number of 20 sections per mouse (n 4 mice) were analyzed and PKH-26 positive cells were counted. Data were expressed as number of PKH-26 positive cells per 105 renal cells. hCB-MSCs were also identied in renal sections stained with mouse anti-human nuclear antigen (HNA) followed by donkey anti-mouse-Cy5 (Vector Laboratories). Samples were counterstained with FITC-labeled lectin WGA and DAPI. The engraftment of hCB-MSCs-PKH-26 positive was also evaluated at 4 days in other organs such as liver, lung, and spleen. Tissue samples were xed as above and sections stained with DAPI. Six sections per mouse (n 3 mice) were analyzed for each organ, and PKH-26 positive cells were counted.

Real Time Reverse Transcription (RT)-PCR of HGF

HGF mRNA expression was determined by real time PCR on kidneys of cisplatin-mice given saline or CB-MSCs. Total RNA was isolated as previously described [41]. Amplication was performed with ABI 7300 Real Time PCR System using SYBR GREEN PCR Master Mix and following specic primers: mouse HGF1 forward 50 -TCAAATGCCAGCCTTGGAA-30 , reverse 50 glyceraldehyde-3-phosGCAAAAAGCTGTGTTCATGGG-30 ; phate dehydrogenase forward 50 -TCATCCCTGCATCCACTGGT30 , reverse 50 -CTGGGATGACCTTGCCCAC-30 . The DDCt technique was used to calculate cDNA content in each sample using the cDNA expression in mouse with vehicle as calibrator.

In Vitro Experiments
For coculture experiments, conuent proximal tubular cell line (HK2; CTR-2190; American Type Culture Collection, Manassas, VA, http://www.atcc.org) was exposed to 8 lM cisplatin for 6 hours in a-MEM, plus 5% FBS. After drug withdrawal, hCBMSCs (5 104 cells per well) were added in Transwell system with HK-2 cells (test medium a-MEM plus 5% FBS). Cell supernatants obtained by cisplatin-treated HK-2 cells alone or in coculture with hCB-MSCs were collected at 2472 hours after cisplatin.

Proteome Assays
Growth factors and inammatory cytokines produced by coculture experiments were quantied by SearchLight proteome array, a multiplexed sandwich ELISA (Pierce-Endogen, Illinois, USA) in conditioned cell supernatants. Supernatants were analyzed by Human Angiogenesis Array one hepatocyte growth factor (HGF) broblast growth factors (FGF) HB-EGF, vascular endothelial growth factor (VEGF) and the Human Inammatory Cytokine one interleukin-1 beta (IL-1B) and tumor necrosis factor-alpha; (TNFa) following manufacturers instructions. ArrayVision Software for data acquisition and management was used following manufacturers instructions. In addition, serum levels of HGF were evaluated in cisplatin-mice given saline (n 10 mice) or hCB-MSCs (n 10 mice) at 4 days using the same multiplexed sandwich ELISA.

Immunohistochemistry
Oxidative Damage. To determine protein nitration of tyrosine
residues, parafn kidney sections (3 lm) were xed with methanol and incubated with 30% H2O2 for 30 minutes and then with 0.3% Triton X-100/PBS (phosphate buffered saline) for 15 minutes. Sections were incubated with primary antibody (rabbit polyclonal anti-nitrotyrosine, 1:2,000; Upstate Biotechnology, Billerica, MA) followed by secondary antibody (biotinylated goat anti-rabbit IgG). Signal was developed using Vectastain ABC kit and 3,30 diaminobenzidine (DAB) reagents (Vector Laboratories). Negative controls were performed omitting the primary antibody. At least 3040 nonoverlapping sequential elds (n 4 mice for each group) were analyzed. Each section was scored for intensity (absent, faint, moderate, intense: 0 to 3).

Statistical Analysis
The results are expressed as mean 6 SE. Statistical analysis was performed using ANOVA followed by Tukey Cicchetti test for multiple comparisons, nonparametric Kruskal-Wallis test, logrank test, or Kaplan Meier as appropriate. The Wilcoxon-based method was used to determine the signicance of differences between independent groups. Statistical signicance level was dened as p < .05.

Apoptosis. Apoptosis was measured by a terminal transferasemediated dUTP nick-end labeling (TUNEL) assay using an in situ cell detection kit (Roche, Mannheim, Germany) followed by counterstaining with Rhodamine-labeled lectin Lens Culinaris Agglutinin (LCA; Vector Laboratories) and DAPI for 15 minutes. Slides were mounted with mounting medium and analyzed by confocal microscopy. Apoptotic nuclei and DAPI-positive cells per eld (n 5 mice for each group) were counted and the results were expressed as TUNEL positive cells per 103 cells.

RESULTS
Akt. Frozen kidney sections, xed in acetone, were incubated
with anti-phosphorylated (p) Akt (Ser 473) antibody (Cell Signaling Technology, Danvers, USA) followed by goat anti rabbit-Cy3 (Jakson Immunoresearch, West Baltimore, Pike). Slides were counterstained with FITC-labeled lectin WGA (Vector Laboratories) and nuclei were stained with DAPI. Thirty elds per mouse (n 3 mice for each group) were analyzed and pAkt-positive tubules were counted. Data were expressed as percentage of pAkt-positive tubules per eld.

Morphology, Differentiation Potential, and Immunophenotype of hCB-MSCs

Eighty human CB units were processed and 54% generated MSC cultures. hCB-MSCs isolated by lineage depletion represented 12% of the initial cell population and grew up to 10 passages maintaining their typically spindle-shaped morphology (Fig. 1A). By exposure to appropriate inductive media, hCB-MSCs differentiated into osteocytes and chondrocytes as indicated by the extended area of mineralization and by secretion of cartilage-specic proteoglycans (Fig. 1B, 1C). At variance, hCB-MSCs did not possess adipogenic potential as also previously described [31, 42, 43].

Proliferation. To determine the number of proliferating cells, formalin-xed parafn sections were stained with a monoclonal anti-proliferating cell nuclear antigen (PCNA) clone PC10 (1:250; Sigma-Aldrich) followed by biotinylated sheep anti-mouse IgG (1:50; Chemicon). PCNA signal was developed by Vectastain www.StemCells.com

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Figure 1. Characterization of human (h) cord blood-mesenchymal stem cells (CB-MSCs). (A): Representative image of hCB-MSCs with typical spindleshaped morphology. Original magnication, 10. (B): Osteogenic differentiation of hCBMSCs is evidenced by Alizarin Red staining with area of mineralization. Original magnication, 20. (C): Proteoglycans stained by Alcian Blue reveals chondrogenic differentiation. Original magnication, 20. (D): Flow cytometric characterization of hCB-MSCs. Representative dot plots showing the expression of CD105, CD44, CD90, CD73, HLA class I, CD146, aSMA of hCB-MSCs at passage 6. The respective isotype control is shown as a red line. Abbreviations: HLA, hyuman leukocyte antigen; aSMA, a-smooth muscle actin; SSEA, stage-specic embryonic antigen.

The expression of cell-surface antigens was evaluated by ow cytometry on all hCB-MSC cultures. As shown in Figure 1D (representative analysis) and Table 1, hCB-MSCs expressed CD105, CD44, CD90, CD73, SSEA4, and HLA class I. hCB-MSCs were weakly positive for CD56, desmin, CK3/12, CK19, and BB9, and negative for HLA class II, NGFR, CXCR4, and VE-Cadherin. These cells also expressed CD146 and alpha SMA (but not CD34, CD45, and NG2), which altogether characterize a multipotent adult stem cell population consistent with a perivascular/pericyte-like phenotype (Fig. 1D; Table 1) [44]. The colocalization of embryonic marker SSEA4 with CD146 was also observed by immunouorescence (Supporting Information Fig. 1). The molecular prole of hCB-MSCs by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the cells expressed some genes including RUNX1 and OCT4 mRNA, markers that characterize the undifferentiated stem cell state (Supporting Information Fig. 2). hCB-MSCs did not express PPAR-2, VE-Cadherin, ABCG2, REX1, FGF4, hTERT, SOX2, PAX7, and renal genes such as PAX2 and GATA3.

Table 1. Immunophenotype of hCB-MSCs

Antigens Percentage of uorescent cells

CD105 CD44 CD90 CD73 SSEA4 HLA cl I CD146 a SMA CD56 DESMIN CK3/12 CK19 BB9

55.3 62.8 90.4 98.8 3.9 96.6 35 53.5 2.1 0.4 1 2.6 0.2

6 6 6 6 6 6 6 6 6 6 6 6 6

37.3 35.1 6.9 1.9 4.1 2.9 18.1 19.6 1.4 0.3 0.7 1.7 0.1

Data are expressed as mean 6 SD. Abbreviation: hCB-MSCs, human cord blood-mesenchymal stem cells.

Therapeutic Effect of hCB-MSCs in Cisplatin-Mice with AKI

The renoprotective potential of hCB-MSCs was investigated in immunodecient mice with cisplatin-induced AKI. Previous experiments [13] indicated that subcutaneous injection of cisplatin (12.7 mg/kg) in NOD-SCID mice caused renal function impairment, as indicated by increased levels of BUN, which peaked at 4 days and stabilized to high values until animal death within 57 days. Here, we tested the effect of i.v. injection of hCB-MSCs or saline in NOD-SCID mice, 1 day after cisplatin treatment, when tubular ultrastructural changes were already present [13]. Renal function and histology were studied at 4 days, the time at which all animals with AKI were still alive. As shown in Figure 2A, hCB-MSC infusion mark-

edly improved renal function, as reected by the signicant (p < .01) reduction of BUN levels at 3 and 4 days in respect to mice given saline. Renal histology of mice with cisplatininduced AKI on day 4 showed focal and severe tubular cell degenerative changes with necrosis and luminal casts (Fig. 2B; Table 2). In kidneys of cisplatin-mice injected with hCBMSCs, renal tubular damage was markedly attenuated as reected by the signicant reduction of numbers of necrotic tubules and casts (Fig. 2B; Table 2). Electron microscopy analysis of renal tissue of cisplatin-treated mice sacriced at 4 days conrmed the presence of necrotic epithelial cells with detachment from the underlying basement membrane, and showed peritubular capillary changes consisting of endothelial cell swelling, cytoplasmic vacuolisation, nuclear degeneration, and detachment, occasionally associated with

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Figure 2. hCB-MSC infusion protects NOD-SCID mice with acute kidney injury (AKI) from renal function impairment and tubular injury. (A): Renal function, assessed as BUN, in cisplatin-treated mice given saline or hCB-MSCs. Data are mean 6 SE.  p < .01 versus day 0; *p < .01 versus saline. (B): Histological changes in kidneys from control and cisplatin-treated mice receiving saline or hCB-MSCs at 4 days. Original magnication, 400. (C): Ultrastructural changes of tubular epithelium and peritubular capillaries in mice given saline or hCB-MSCs 4 days after cisplatin and effect of treatment with hCB-MSCs. Note the presence of tubular and endothelial cell damage with PMN cell inltration in cisplatin-treated mouse given saline. Original magnication, 2,800. Abbreviations: BUN, blood urea nitrogen; hCB-MSCs, human cord blood-mesenchymal stem cells; PMN, polymorphonuclear.

polymorphonuclear (PMN) cell inltration (Fig. 2C). At variance, in the kidneys of cisplatin-mice infused with hCBMSCs, peritubular capillary changes were either mild or absent (Fig. 2C). Kidneys of mice sacriced at 24 hours showed no signicant abnormalities of peritubular microvascular structures (data not shown). No sign of maldifferentiation of hCB-MSCs into other cell types of non-renal origin or adipocytes, as instead previously documented for rat BM-MSCs [40], was observed in renal tissue of cisplatin-mice at 40 days.

Table 2. Effect of hCB-MSCs on renal histological changes at 4 days

Casts (no of casts/HPF) Tubular necrosis (no/HPF)

Control Cisplatin saline Cisplatin hCB-MSCs

0 2.98 6 0.8 0.13 6 0.04*

0 1.79 6 0.4 0.09 6 0.03*

Distribution of hCB-MSCs Infused in Cisplatin-Mice

To evaluate the engraftment of hCB-MSCs in the renal parenchyma of mice with AKI, stem cells were labeled with PKH26a red uorescence cell membrane linkerbefore their in vivo infusion. In renal tissues of cisplatin-treated mice at 4 days, PKH-26-positive cells were 2 6 0.4/105 renal cells, and they were predominantly found in peritubular areas, and to a very low extent in the context of tubular epithelium and glomeruli (Fig. 3A, left). The percentage of PKH-26 positive CB-MSCs (expressed as number of PKH-26 cells in each www.StemCells.com

Data are mean 6 SE. *p < .01 versus Cisplatin saline. Abbreviations: hCB-MSCs, human cord blood-mesenchymal stem cells; HPF: high-power eld.

compartment per total PKH-26 cells) averaged 83% 6 11% in peritubular areas, 5% 6 5% in tubules, and 12% 6 12% in glomeruli. The human origin of CB-MSCs was conrmed in murine renal tissue by the positive staining for the HNA (Fig. 3A, right). In other organs, including liver, lung, and spleen, hCB-MSCs positive for PKH-26 were rare or absent.

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.004; and hBM-MSCs, p < .05 vs. untreated cisplatin-mice). By comparing renal function of mice infused with hCB-MSCs or hBM-MSCs, we found that serum BUN levels in cisplatinmice treated with hCB-MSCs at 4 days were numerically lower than those found in hBM-MSC-treated mice although a statistical signicance was not achieved (hCB-MSCs, 58 6 7 and hBM-MSCs, 76 6 8 vs. saline, 122 6 7 mg/dl) (hCBMSCs, p < .01 and hBM-MSCs, p < .01 vs. saline).

hCB-MSC Infusion Improves Survival of AKI Mice

We investigated whether i.v. infusion of hCB-MSCs in NODSCID mice could effect survival, the key clinical outcome in AKI. Eighty-six percent of cisplatin-mice given saline died within 7 days, reaching 100% after 2 days (Fig. 3B). hCBMSC infusion remarkably prolonged animal survival (p < .001) to the extent that 86% of cisplatin-mice were alive at 9 days when all mice died in the saline group. The percentage of living animals given hCB-MSCs remained unchanged until 14 days, when the experiment was stopped. The efcacy of hCB-MSCs on lifespan of AKI mice was compared with that of hBM-MSCs, previously described to decrease animal mortality in the same experimental model [13]. As shown in Table 3, infusion of hCB-MSCs improved animal survival to a more signicant extent than hBM-MSCs in respect to cisplatin-mice given saline (hCB-MSCs, p <

hCB-MSCs Inhibit Cisplatin-Induced Tubular Oxidative Damage and Apoptosis

Oxidative damage caused by reactive oxygen species has been implicated in the pathogenesis of cisplatin-induced renal failure [45]. Peroxynitrite, the reaction product of nitric oxide with superoxide anion, has been taken as key oxidant species involved in the direct nitration of tyrosine residues causing protein oxidation [46]. In cisplatin-mice with AKI, the expression of nitrotyrosine was signicantly (p < .05) increased at 4 days in tubules of both the cortex and the medulla in respect to control mice (Fig. 4A; Table 4). A marked reduction of nitrotyrosine staining at tubular levels was found when cisplatin-mice were treated with hCB-MSCs (Fig. 4A; Table 4), indicating that human stem cells protect renal tissues from oxidative damage. As in vitro and in vivo models of cisplatin-induced toxicity have shown that apoptosis represents one of the major causes of tubular cell loss [47], we studied whether hCBMSC treatment could exert an anti-apoptotic action on renal cells in mice with AKI. Cells positive for TUNEL staining were quantied in kidney sections at day 4 by confocal laser scanning microscopy. A signicant increase (p < .01) in the number of apoptotic cells was observed in renal tissue of mice treated with cisplatin with respect to control mice (Fig. 4B). hCB-MSC infusion resulted in a signicant (p < .01) reduction of TUNEL-positive cells as compared with cisplatin-mice given saline (Fig. 4B), thereby suggesting a prosurvival effect of the stem cell therapy in mice with AKI.

Figure 3. hCB-MSCs engraft the kidney in mice with acute kidney injury (AKI). (A): Representative micrograph of kidney tissue from cisplatin-treated mouse injected with PKH-26-labeled hCB-MSCs at 4 days (left). PKH-26 uorescent CB-MSCs (red) was localized in peritubular area (arrow). Original magnication, 630. Micrograph showing hCB-MSCs in a kidney section of cisplatin-treated mouse at 4 days stained for human antigen HNA (red, right). Original magnication, 630. The sections were stained with FITC-labeled lectin wheat germ agglutinin (green), and 40 ,60 -diamidino-2-phenylindole dihydrochloride hydrate for nuclei (blue). (B): hCB-MSC treatment prolongs survival in mice with AKI. All mice receiving saline died within 9 days. At this time, 86% of NOD-SCID mice given hCB-MSCs survived. p < .001 versus cisplatin saline. Abbreviation: hCB-MSCs, human cord blood-mesenchymal stem cells.

hCB-MSCs Induce Akt Phosphorylation and Enhance Tubular Cell Proliferation in Mice with AKI
Growing evidence suggests that the serine-threonine kinase Akt protects cells from apoptosis as a key enzyme in survival signals [48]. Expression of tubular pAkt transiently increased in renal tissue of mice 24 hours after cisplatin injection (data not shown) and then declined at 4 days with respect to control animals (Fig. 4C). hCB-MSC injection signicantly increased the number of tubuli positive for pAkt at 4 days (Fig. 4C). Tubular cell proliferation as an index of renal regeneration in the context of acute renal failure was also studied. As reported in Figure 4D, tubular cell proliferation, highlighted by staining and quantication of PCNA, slightly increased in cisplatin mice given saline as compared with controls, indicating a spontaneous weak induction of the reparative process.

Table 3. Comparison between hCB-MSCs and hBM-MSCs on survival of cisplatin-treated mice

Survival (%) 0 day 4 days 5 days 7 days 9 days 14 days

Cisplatin saline Cisplatin hCB-MSCs Cisplatin hBM-MSCs

100 100 100

100 100 100

50 100 90

0 80 30

0 70 30

0 70* 30**

**p < .004 hCB-MSCs versus Cisplatin saline.  p < .05 hBM-MSCs versus Cisplatin saline (Kaplan-Meier analysis and Log-Rank test, 22, 97). Abbreviations: hBM-MSCs, human bone marrow-mesenchymal stem cells; hCB-MSCs, human cord blood-mesenchymal stem cells.

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Figure 4. hCB-MSC treatment reduces renal oxidative damage and apoptosis, and enhances tubular cell proliferation and Akt phosphorylation in mice with AKI at 4 days. (A): Representative images of nitrotyrosine staining (dark brown), a marker of peroxynitrite, in renal tissue of control and cisplatin-treated mice receiving saline or hCB-MSCs. Original magnication, 400. (B): TUNEL-positive cells quantied in kidney sections of control mice, cisplatin-treated mice given saline or hCB-MSCs.  p < .01 versus control; *p < .01 versus cisplatin saline. Representative images of kidney sections labeled with TUNEL (green), rhodamine-labeled lectin Lens Culinaris Agglutinin (red) and 40 ,60 -diamidino-2-phenylindole dihydrochloride hydrate (DAPI; blue). Original magnication, 630. (C): Percentage of pAkt-positive tubules in control mice, cisplatin-treated mice receiving saline or hCB-MSCs.  p < .01 versus control; *p < .01 versus cisplatin saline. Representative images of pAkt staining (red) with uorescein isocyanate-labeled lectin Wheat Germ Agglutinin (green) and DAPI (blue) in kidney sections of control, cisplatin-treated mice given saline, or hCB-MSCs. Original magnication, 630. (D): Quantication of PCNA-positive tubular cells. p < .01 versus control; *p < .05 versus cisplatin saline. Abbreviations: hCB-MSCs, human cord blood-mesenchymal stem cells; PCNA, proliferating cell nuclear antigen.

Importantly, the treatment with hCB-MSCs strongly induced cell division, thus strengthening the meaning of a powerful induction of tissue recovery.

Table 4. Effect of hCB-MSCs on nitrotyrosine expression

Tubuli of cortex (score) Tubuli of medulla (score)

Growth Factor and Inammatory Cytokine Expression

The capability of hCB-MSCs to create a proregenerative environment instrumental to promote tissue repair was explored in vitro. We carried out experiments by coculturing hCB-MSCs with cisplatin-treated proximal tubular cells (HK-2 cells) in a transwell system for 24 hours. The production of growth www.StemCells.com

Control Cisplatin saline Cisplatin hCB-MSCs



0.60 6 0.06 1.69 6 0.10  0.98 6 0.21*

0.39 6 0.03 1.29 6 0.08 0.54 6 0.11*

Data are mean 6 SE. p < .05 versus Control; *p < .05 versus Cisplatin saline. Abbreviation: hCB-MSCs, human cord blood-mesenchymal stem cells.

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Figure 5. Effect of hCB-MSCs cocultured with cisplatin-damaged HK-2: human kidney-2 cells on the production of IL1b and TNFa: tumor necrosis factor-alpha. HK2 were incubated with cisplatin (8 lM) for 6 hours, and after drug withdrawal they were cocultured with hCB-MSCs for 2472 hours.*p < .05 versus HK-2 cells cisplatin. Abbreviations: hCB-MSCs, human cord blood-mesenchymal stem cells; HK2, human kidney-2; IL1b, interleukin-1 beta; TNFa, tumor necrosis factor-alpha.

factors including FGF, HB-EGF, VEGF, and particularly HGF signicantly (p < .05) increased in the supernatants of cell cocultures with respect to those of damaged HK-2 cells (FGF: 2-, HB-EGF: 7-, VEGF: 5-, and HGF: 12-fold increase). Given the robust expression of HGF in in vitro experimental setting, we further focused on the expression of this growth factor in vivo. Serum levels of HGF were signicantly increased at 4 days in cisplatin-mice receiving hCB-MSCs in respect to mice given saline (779 6 120 vs. 382 6 63 pg/ml, p < .05). In parallel, kidneys of animals treated with hCBMSCs showed a signicant upregulation of HGF mRNA expression at 4 days (hCB-MSCs, 1.8 6 0.1-fold increase vs. saline, 1 6 0.03, p < .0001). Cisplatin-induced renal toxicity is mediated by inammatory cytokines, including IL-1b and TNFa, produced by renal parenchymal cells [49, 50]. To determine whether hCBMSCs, besides their regenerative ability, possess anti-inammatory properties, proximal tubular cells pre-exposed to cisplatin were cocultured with hCB-MSCs for different time intervals. As shown in Figure 5, the release of IL-1b and TNFa by proximal tubular cells exposed to cisplatin signicantly increased at 24 and 72 hours. When hCB-MSCs were added to the coculture system, a signicant inhibition of cytokine production was observed during time (Fig. 5).

DISCUSSION
The present study provides for the rst time evidence that hMSCs derived from CB, intravenously delivered to immunodecient mice with AKI, protect animals from renal function impairment and remarkably prolong lifespan. In agreement with our previous observations that hMSCs of BM origin are of value in the treatment of experimental AKI [13], these ndings identify a suitable cell population for the possible use in future studies of cell therapy for human AKI. Data that stem cell transplantation can prolong animal survival have been reported before for BM-derived cells [13]. Here, however, we found that the effect of hCB-MSCs is considerably stronger. The reasons for this difference still remain to be elucidated. A possible explanation may rest on the evidence that hCB-MSCs share with h. BM-MSCs similar expression of a

considerable number of transcripts but hCB-MSCs express angiogenic and matrix remodeling genes at a higher extent [32]. The use of MSCs of CB origin may be helpful in patients for whom the availability of autologous functional stem cells is limited. Patients given cisplatin might have had previous chemotherapy which could compromise the availability of mesenchymal cells in BM [51]. For these patients, hCB-MSCs can be an interesting option. hCB-MSCs are a valuable source of stem cells for cellular therapy in terms of access and supply, large in vitro expansion capacity, and differentiation potential [19, 21, 31]. In the last years, different approaches of hCB-MSC isolation have been developed. The rate of success has been increased from 25% of efciency in 2001 [52] to 54% in 2009, as achieved by the present study. The noninvasive nature of this collection represents an advantage over any other types of stem cells [53]. Phenotypes, cell cycle status, and surface markers of hCB-MSCs are almost identical with BM-MSCs [43, 54]. No differences in the osteogenic and chondrogenic differentiation capacity were detected between hBM-MSCs and hCB-MSCs, whereas the present article also conrms the absence of adipogenic differentiation by hCB-MSCs only [31, 42, 43]. Moreover, hCB-MSCs have many advantages partly related to the immaturity of newborn cells compared with adult stem cells and their being prone to escape immune rejection [21, 35, 36]. For these reasons, human umbilical CB in the last years has been banked and used as a source of marrow repopulating stem cells in patients with BM decit or inborn errors of metabolism [21, 3537] resulting safe and efcacious. Once established the capacity of hCB-MSCs to repair renal injury, we then addressed the mechanisms possibly implicated in the renal reparative processes. hCB-MSCs were able to engraft and survive in kidneys of NOD-SCID mice previously injected with cisplatin, whereas rare or no cells were found in other organs including liver, lung, and spleen. In the renal tissue, the cells almost exclusively localized in the peritubular areas, not in the context of the tubular epithelial lining, which would reasonably exclude that hCB-MSCs acted by transdifferentiating into proximal tubular cells. These data are in support of a paracrine action of hCB-MSCs once engrafted in the damaged kidney, as already described for BM-derived stem cells in experimental AKI by other and our laboratories [12, 23, 24]. This may happen via the local release of soluble factors [12, 23] and/or via the stimulation of target cells to produce growth factors with regenerative potential for renal cells. Relevant to the former interpretation are data that hCB-MSCs in culture constitutively produce prosurvival, anti-inammatory, and mitogenic proteins [34, 55]. Of interest, the remarkable amounts of growth factors as FGF, HB-EGF, VEGF, and particularly HGF, produced by hCBMSCs in coculture with cisplatin-damaged tubular cells imply a mechanism whereby the hCB-MSCs homing to the damaged kidney create a regenerative environment for tubular repair. The robust expression of HGF in renal tissue of cisplatinmice given hCB-MSCs translates in vitro nding into the in vivo condition. Hypoxic conditions and inammatory cytokines, mediators of AKI, can enhance the expression of growth factors with mitogenic and anti-inammatory action by MSCs [34, 56]. Our present data are in support of the paradigm that locally delivered growth factors by stem cells and/ or tubular cells in response to CB-MSCs could act in concert to mitigate renal injury and hasten repair. The combined actions of several factors to simultaneously modulate different pathological pathways of disease, that is, inammation and apoptosis, would result in a superior renoprotection than any single growth factor treatment, which potentially bears systemic side effects [57].

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Next, we addressed the intracellular pathways of renoprotection conferred by hCB-MSCs. Renal tubular toxicity of cisplatin manifested with oxidative damage and cell apoptosis, both of which were ameliorated to a considerable extent by hCB-MSC treatment. The present data are consistent with the results of previous studies on reactive oxygen species (ROS) as key determinant of cisplatin-induced apoptosis [58], and on the favorable effect of antioxidants and ROS scavengers in limiting programmed cell death [58] and reducing tubular cell oxidative damage. To nd a link between the capacity of stem cells to counteract apoptosis and release prosurvival factors, one might speculate that growth factors prevent oxidative stress in damaged tissues leading to cell protection through their ability of normalizing antioxidant enzyme activity and mitochondrial function as membrane potential and oxygen consumption [59]. Thus, HGF therapy enhanced survival of the cells subjected to the oxidative stress [60, 61] in experimental model of myocardial infarction [62]. On the other hand, the above growth factor together with VEGF also have the ability to activate the prosurvival factor Akt [63], a kinase that plays a critical role as mediator of the phosphoinositide-3 kinase (PI3K)-survival signals [48]. pAkt counteracts cell apoptosis by phosphorylating and inhibiting the proapoptotic member of the Bcl-2 family, Bad [64], and the mitochondrial caspase-9 [65, 66]. Recent ndings have documented that blockade of PI3K-c-Akt pathway markedly accelerated renal tubular cell apoptosis and death leading to poor prognosis for mice with cisplatin-induced AKI [67]. The relevance of Akt in renal repair was conrmed by our data showing that in renal tissue of cisplatin-mice, phosphorylation of Akt, markedly decreased at 4 days, the time point with higher tubular damage and apoptosis. Conversely, treatment with hCB-MSCs signicantly increased pAkt-positive tubuli and possibly activated downstream targets known to modulate cell proliferation and survival [48]. Akt activation at tubular level was indeed associated with a marked induction of proximal tubular cell proliferation as evidenced by the high number of cells positive for PCNA staining at 4 days, after hCB-MSC injection. To explore the possible anti-inammatory action of hCBMSCs, here we have documented that cultured proximal tubular cells exposed to cisplatin enhanced the production of cytokines as TNFa and IL1b which was markedly reduced by coculture with hCB-MSCs. Previous studies have convincingly described that these proinammatory cytokines are upregulated in experimental and human AKI [49, 50, 68] and are described to perturb peritubular endothelial cells to overexpress E-selectin and inter-cellular adhesion molecule-

1 (ICAM-1) thus favoring inammatory leukocyte migration [69, 70]. Present in vivo ndings suggest that hCB-MSC therapy limited PMN leukocyte inltration in peritubular capillaries and reduced microvascular peritubular damage that follows cisplatin-induced tubular injury [13] and provide further evidence that hCB-MSCs contribute to kidney regeneration by virtue of their anti-inammatory properties.

CONCLUSION
In conclusion, in a murine model of AKI, hCB-MSC treatment promotes kidney regeneration and prolongs survival better than any other cellular approach attempted so far. These effects appear to be mediated by a paracrine action of hCBMSCs on tubular cells involving lowering oxidative stress, apoptosis, and inammation. These data indicate that hCBMSCs have to be considered as one possible future option for cellular therapy of AKI in humans.

ACKNOWLEDGMENTS
We thank Daniela Corna and Daniela Rottoli for excellent technical assistance. We are indebted to Dr. Annalisa Perna for helping with statistical analysis. This study was supported by grants from Fondazione Cariplo (Milano, Italy; Grant 2007-5549), Fondaazione Il Sangue; the Ministero della Salute (Progetto a concorso 2009; Ricerca corrente 2008 e 2009, Ex. Art. 56), Istituto ` Superiore di Sanita (Malattie Neurodegenerative 2006), Sixth FP Thercord (contract LHSB-CT-2006-018817), Seventh FP CASCADE (Cultivated Adult Stem Cells as Alternative for Damaged Tissues-HEALTH-F5-2009-223236). Foundation Novussanguis and Foundation Jerom Lejeune. Part of the research leading to these results has received funding from the European Community under the European Communitys Seventh Framework Programme (FP7/2007-2013), grant number 223007, STAR-TREK project. C.R. is a recipient of a fellowship from Fondazione Aiuti per la Ricerca sulle Malattie Rare (ARMR), Bergamo, Italy.

DISCLOSURE

OF OF

POTENTIAL CONFLICTS INTEREST

The authors indicate no potential conicts of interest.

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